JP3641013B2 - Novel cell adhesion inhibitors Macrospheride A and B and process for producing them - Google Patents

Description

【００１２】
III ．生理的、生態的性状
１）本菌株の最適生育条件はＧＰ液体培地においてｐＨ４〜１０、最適生育温度はＹｐＳｓ寒天培地において１５〜３０℃である。
２）本菌株の生育範囲はＧＰ液体培地においてｐＨ３〜１０、生育温度はＹｐＳｓ寒天培地において７〜３５℃である。
３）好気性、嫌気性の区別：好気性
【００１３】
以上の形態的観察および培養、生理的性状に基づき既知菌株との比較を試みた結果、本菌株は分生子果不完全菌綱、ミクロスフェロプシス（Ｍｉｃｒｏｓｐｈａｅｒｏｐｓｉｓ）属に属する菌株と同定し、ミクロスフェロプシス エスピー（Ｍｉｃｒｏｓｐｈａｅｒｏｐｓｉｓ ｓｐ．）ＦＯ−５０５０と命名した。なお、本菌株は、ミクロスフェロプシス エスピー ＦＯ−５０５０として工業技術院生命工学工業技術研究所に寄託されている。寄託日は平成７年２月２０日で、受託番号はＦＥＲＭ Ｐ−１４７７６である。
【００１４】
本発明の好ましい菌株として、マクロスフェライド類生産菌を説明したが、糸状菌の一般的性状としての菌学上の性状はきわめて変異し易く、一定したものではなく、自然的にあるいは通常行われる紫外線照射、Ｘ線照射または変異誘導体、例えばＮ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアジン、エチルメタンスルホネートなどを用いる人工的変異手段により変異することは周知の事実であり、このような人工的変異株は勿論、自然変異株も含め、ミクロスフェロプシス属に属し、マクロスフェライド類を生産する能力を有する菌株はすべて本発明に使用することができる。
【００１５】
また、細胞融合、遺伝子操作などの細胞工学的に変異させた菌株も本発明のマクロスフェライド類生産菌として包含される。本発明においては、先ずミクロスフェロプシス属に属するマクロスフェライド類生産菌が、適当な培地に培養される。本菌の培養においては、通常の糸状菌の培養方法が一般に適用される。培地としては微生物が同化し得る炭素源、消化し得る窒素源、さらに必要に応じ無機塩などを含有させた栄養培地が用いられる。
【００１６】
同化し得る炭素源としては、ブドウ糖、糖密、澱粉、デキストリン、セルロース、コーン・スティープ・リカー、グリセリン、有機酸などが単独または組み合わせて用いられる。消化し得る窒素源としては、例えば市販されているペプトン、肉エキス、酵母エキス、乾燥酵母、大豆粉、コーン・スティープ・リカー、綿実粉、カゼイン、大豆蛋白分解物、アミノ酸、尿素などの有機窒素源、硝酸塩、アンモニウム塩などの無機窒素化合物が単独または組み合わせて用いられる。
【００１７】
更に、必要に応じてナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩、リン酸塩などの無機塩類が添加される。更に、培地には、必要に応じて、本菌の生育やマクロスフェライド類の生産を促進する微量栄養素、発育促進物質、前駆物質を適当に添加してもよい。
【００１８】
培養は通常振とうまたは通気攪拌培養などの好気的条件下で行うのがよい。工業的には深部通気攪拌培養が好ましい。培地のｐＨは中性付近で培養を行うのが好ましい。培養温度は２０〜３７℃でも行い得るが、通常は２４〜３０℃、好ましくは２７℃付近に保つのがよい。培養時間は、液体培養の場合、通常３〜６日培養を行うと、マクロスフェライド類が生成蓄積される。好ましくは培養中のマクロスフェライド類の蓄積量が最大に達したときに培養を終了すればよい。
【００１９】
これらの培養組成、培地の液性、培養温度、攪拌速度、通気量などの培養条件は使用する菌株の種類や外部の条件などに応じて好ましい結果が得られるように適宜調節、選択されることはいうまでもない。液体培養において発泡があるときは、シリコン油、植物油、界面活性剤などの消泡剤を適宜使用してもよい。
【００２０】
このようにして得られた培養物中に蓄積された活性物質は培養濾液または培養菌体中に含まれている。培養濾液を必要に応じて濾過補助剤、例えばセライト、ハイフロースーパーセル等を加えて濾過するか、または遠心分離して培養濾液と菌体とに分離し、培養濾液と菌体との有機溶媒抽出物を濃縮したものの混合物からマクロスフェライド類を採取するのが有利である。
【００２１】
また、培養濾液及び菌体を分離しないで、そのまま非親水性有機溶媒により抽出することができる。また、マクロスフェライド類の含有量が培養濾液と菌体のどちらかに極端に多いときはその多い方から抽出してもよい。培養菌体からマクロスフェライド類を分離精製するためには、通常、培養ろ液と菌体の有機溶媒抽出物を濃縮した物の混合物またはそれぞれを非浸水性溶媒、例えば酢酸エチル、クロロホルムで抽出することにより、マクロスフェライド類が有機溶媒に転溶される。
【００２２】
このようにして得られた有機溶媒層は、必要に応じ種々の脱水剤、たとえば無水硫酸ナトリウム、ビーズゲルなどを加えて脱水された後、減圧下で有機溶媒が留去される。この濃縮操作においてマクロスフェライド類は安定な物質であるが、通常加熱温度を５０℃以下となるように行うのが好ましい。残渣にヘキサン、石油エーテルなどの有機溶媒を加えてマクロスフェライド類を沈澱させることができる。
【００２３】
得られた沈澱物は数回ヘキサンなどで洗浄後、吸引濾過または遠心分離によりマクロスフェライド類の粗製物を採取することができる。この粗製物をさらに精製するためには、マクロスフェライド類と混雑物との溶解度の差や混じり合わない二液相関の分配の差や各種吸着担体に対する吸着力の差を利用した多くの手段が可能であるが、特にクロマトグラフィーはマクロスフェライド類の精製に有効な方法である。
【００２４】
マクロスフェライド類の精製に有効なクロマトグラフィーとしては、シリカゲル、アルミナ、活性炭セルロース、ヒドロキシアパタイトＨＰ−２０などの吸着樹脂などによる吸着クロマトグラフィー、シラン化シリカゲル、オクタデシルシラン化シリカゲルなどを用いる逆相分配クロマトグラフィー、セファデックスＬＨ−２０、トヨパール（商品名、東ソー社製）などを用いる分子ふるいにもとずくゲル濾過クロマトグラフィーなどが挙げられる。
【００２５】
マクロスフェライド類は、これらのクロマトグラフィーや電気泳動、向流分配、限外濾過などの手段を、単独あるいは任意の順序にて組み合わせまたは反復して用いることにより、分離精製することができる。例えば上記の粗製物を少量のクロロホルムに溶かし、シリカゲルに吸着させ、クロロホルム−アセトン系混合溶液を用いてカラムクロマトグラフィーを行い、その活性画分を減圧濃縮後、少量のメタノールに溶かし、これをメタノールで分子ふるいにもとずくゲル濾過クロマトグラフィーを行うことによりマクロスフェライドを分離精製することができる。
【００２６】
次に、本発明マクロスフェライド類の理化学的性質について以下に述べる。
〔１〕細胞接着阻害剤マクロスフェライドＡ
（１）性状：無色針状結晶
（２）元素組成：Ｃ₁₆Ｈ₂₂Ｏ₈ （高分解能マススペクトルによる）
（３）融点：１４１〜１４２℃（クロロホルム）
（４）比旋光度：〔α〕_D ²³＋８４．１゜（ｃ＝０．５９，ＭｅＯＨ）
（５）分子量：３４２（高速原子衝撃（ＦＡＢ）マススペクトルによる）
【００２７】
（６）紫外線吸収スペクトル：メタノール中で測定した紫外部吸収スペクトルは図１に示すとおりであり、２０７（ｌｏｇ ε＝４．２１）ｎｍ付近に特徴的な吸収極大を示す
（７）赤外線吸収スペクトル：ＫＢｒ法で測定した赤外線吸収スペクトルは図２に示すとおりであり、１７１３，３４２８ｃｍ^-1に吸収帯を有する
（８）溶剤に対する溶解性：アセトン、酢酸エチル、エチルエーテル、ヘキサン、メタノール、エタノール、クロロホルムに可溶、水に難溶
【００２８】
（９）呈色反応：硫酸、リンモリブデン酸、ヨウ素、Ｐ−アニスアルデヒドに陽性
（１０）核磁気共鳴スペクトル：日本電子ＥＸ−２７０ＮＭＲスペクトロメータを用いて測定した ¹Ｈ−ＮＭＲスペクトル（重クロロホルム溶液中、２７０ＭＨｚ）および¹³Ｃ−ＮＭＲスペクトル（重クロロホルム溶液中、６７．８ＭＨｚ）はそれぞれ図３及び図４に示すとおりである。また、水素および炭素の化学シフトは下記表２に示す通りである。
【００２９】
【表２】

【００３０】
〔２〕細胞接着阻害剤マクロスフェライドＢ
（１）性状：無色板状結晶
（２）元素組成：Ｃ₁₆Ｈ₂₀Ｏ₈ （高分解能マススペクトルによる）
（３）融点：１４８〜１５０゜（クロロホルム）
（４）比旋光度：〔α〕_D ²³＋４．１゜（ｃ＝０．９８５，ＭｅＯＨ）
（５）分子量：３４０（高速原子衝撃（ＦＡＢ）マススペクトルによる）
【００３１】
（６）紫外線吸収スペクトル：メタノール中で測定した紫外部吸収スペクトルは図５に示すとおりであり、２１０（ｌｏｇ ε＝４．１５）ｎｍ付近に特徴的な吸収極大を示す
（７）赤外線吸収スペクトル：ＫＢｒ法で測定した赤外線吸収スペクトルは図６に示すとおりであり、３４５８，１７４３，１７２６，１７０５ｃｍ^-1に吸収帯を有する
【００３２】
（８）溶剤に対する溶解性：アセトン、酢酸エチル、エチルエーテル、ヘキサン、メタノール、エタノール、クロロホルムに可溶、水に難溶
（９）呈色反応：硫酸、リンモリブデン酸、ヨウ素、Ｐ−アニスアルデヒドに陽性
【００３３】
（１０）核磁気共鳴スペクトル：日本電子ＥＸ−２７０ＮＭＲスペクトロメータを用いて測定した ¹Ｈ−ＮＭＲスペクトル（重クロロホルム溶液中、２７０ＭＨｚ）および¹³Ｃ−ＮＭＲスペクトル（重クロロホルム溶液中、６７．８ＭＨｚ）はそれぞれ図７及び図８に示すとおりである。また、水素および炭素の化学シフトは下記表３に示す通りである。
【００３４】
【表３】

【００３５】
ヒト血管内皮細胞を用いた細胞接着阻害能の評価
ヒト臍帯静脈血管内皮細胞（ＨＵＶＥＣ）に対するヒト骨髄性白血病細胞（ＨＬ−６０）の結合能を指標として細胞接着阻害活性を試験した。
１０％ウシ胎児血清（ＦＢＳ）、ヘパリン１００Ｕ／ｍl 、ＥＣＧＦ５０μｇ／ｍl を含むＭＣＤＢ１０７培地に懸濁したＨＵＶＥＣを２×１０⁴ 個を９６穴のマイクロプレートの各穴に播き、ＣＯ₂ インキュベータ内で３７℃で２４時間培養する。
【００３６】
次いで１０ｎｇの腫瘍壊死因子（ＴＮＦ）を添加してＨＵＶＥＣを刺激した。５時間後に検体ならびに蛍光標識ＨＬ−６０を加え、３０分間インキュベートした。非接着細胞を除去した後、各穴の蛍光強度を測定することにより細胞接着率を算出した、その結果は表４に示した通りである。
【００３７】
【表４】

【００３８】
【発明の効果】
以上のとおり、本発明によるマクロスフェライドＡ及びＢは、細胞接着が深く関与すると考えられる疾患、例えば癌転移、あるいは関節炎などの慢性炎症や喘息、アレルギー疾患等に対しての効果が期待される。
【００３９】
【実施例】
次に実施例を挙げて本発明を具体的に説明するが、本発明はこれのみに限定されるものではない。
試験管（φ２×２０ｃｍ）にグルコース２．０％、酵母エキス０．２％、硫酸マグネシウム・７水和物０．０５％、ポリペプトン０．５％、リン酸二水素カリウム０．１％、寒天０．１％（ｐＨ５．６〜５．８）、１０ｍl を滅菌し、これに酵母エキス０．４％、可溶性デンプン１．５％、リン酸−水素カリウム０．１％、硫酸マグネシウム・７水和物０．０５％、寒天２．０％（ｐＨ６．０）を含む寒天斜面培地上に２７℃、６日間培養したミクロスフェロプシス エスピー ＦＯ−５０５０（ＦＥＲＭ Ｐ−１４７７６）の斜面培養から一白金耳を接種し、２７℃で７２時間振とう培養して種母を得た。
【００４０】
次に、５００ｍl 容三角フラスコに可溶性デンプン３．０％、グリセロール１．０％、キナコ２．０％、乾燥酵母０．３％、塩化カリウム０．３％、炭酸カルシウム０．２％、硫酸マグネシウム・７水和物０．０５％、リン酸二水素カリウム０．０５％を含む液体生産培地（ｐＨ６．５）１００ｍl （２０本）を仕込み滅菌した後、上記の方法で得られた種母１．０ｍl を上記三角フラスコに無菌的に移植し、毎分２２０回転するローターリシェーカーにより２７℃で１２０時間振とう培養を行った。
【００４１】
得られた培養液を液体画分と菌体画分とに分け、液体画分については、酢酸エチルを加えて充分攪拌した後、分液操作により酢酸エチル抽出画分を得た。菌体画分については、これにメタノールを加え、充分攪拌した後、得られたメタノール画分を減圧濃縮してメタノールを留去後、酢酸エチルにより抽出した。液体および菌体の両画分より得た酢酸エチル画分を合わせ、減圧濃縮操作により酢酸エチル抽出画分３７９．４ｍｇを得た。
【００４２】
この酢酸エチル抽出画分を薄層シリカゲル（２０×２０ｃｍ、厚さ０．５ｍｍ、メルク社製）二枚に吸着せしめ、クロロホルム−メタノール混合溶媒（９：１）で展開した。Ｒｆ値が０．５付近で、紫外線ランプ（２５４，３６６ｎｍ）照射下で橙黄色の蛍光を示すバンドを分画し、クロロホルム−メタノール混合溶媒（１：１）で溶出した。得られた溶出画分を減圧濃縮してマクロスフェライド類を含む１８．３ｍｇの粗物質Ｉを得た。
【００４３】
この粗物質Ｉを少量のメタノールに溶解し、高速液体クロマトグラフィー（センシューパック・ペガシル、φ２０×２５０ｍｍ）にかけ、４０％アセトニトリル溶液を移動相として活性画分を分取することによりマクロスフェライドＡの純品１０．２ｍｇ及びマクロスフェライドＢの純品５．２ｍｇをそれぞれ得た。
【図面の簡単な説明】
【図１】マクロスフェライドＡの紫外線吸収スペクトルである。
【図２】マクロスフェライドＡの赤外線吸収スペクトルである。
【図３】マクロスフェライドＡの ¹Ｈ−核磁気共鳴スペクトルである。
【図４】マクロスフェライドＡの¹³Ｃ−核磁気共鳴スペクトルである。
【図５】マクロスフェライドＢの紫外線吸収スペクトルである。
【図６】マクロスフェライドＢの赤外線吸収スペクトルである。
【図７】マクロスフェライドＢの ¹Ｈ−核磁気共鳴スペクトルである。
【図８】マクロスフェライドＢの¹³Ｃ−核磁気共鳴スペクトルである。[0001]
[Industrial application fields]
The present invention relates to novel intercellular adhesion inhibitors Macrospheride A and B that inhibit adhesion factors involved in adhesion between human umbilical vein endothelial cells (HUVEC) and human myeloid leukemia (HL-60) cells, and their production. Regarding the law.
[0002]
[Prior art]
Cancer treatment methods are roughly classified into surgical therapy, radiation therapy, and chemotherapy. With the improvement of the techniques of these methods today or the development of new anticancer agents, the cancer treatment rate has reached about 50%. Gastric cancer, uterine cancer, etc. have been prepared for mass screening and regular screening, and the mortality rate has decreased, but recently, due to changes in eating habits and lifestyle or aging, lung cancer, liver cancer, colon cancer, Although the number of prostate cancers is increasing at present, effective chemotherapeutic agents for these have not yet been satisfactory.
[0003]
[Problems to be solved by the invention]
Thus, the importance of the patient's quality of life (hereinafter referred to as QOL) is now attracting attention as the cancer treatment rate exceeds 50%. However, since anticancer drugs have been developed with the indication of direct cell killing action against cancer cells, they are highly effective but have strong side effects. Therefore, in order to prevent not only treatment but also recurrence and poor prognosis after surgery, development of a chemotherapeutic agent having a new mechanism of action different from the conventional one has been strongly desired.
[0004]
Adhesion of cancer cells to vascular endothelial cells is considered to play an important role in cancer metastasis, and adhesion inhibition is expected as a cancer metastasis inhibitor. Akiyama, S .; K. Et al. Biol. Chem. 264, 18011 to 18018 (1989).
[0005]
Accordingly, the present invention provides novel cell adhesion inhibitors Macrospheride A and B having a new mechanism of action of cell adhesion inhibition, and further, the present invention provides a conidial fruit and fruit incomplete fungus class, microspheres. A microorganism having the ability to produce Macrospherides A and B belonging to the genus Psys is cultured in a medium to accumulate Macrospherides A and B in the culture, and Macrospherides A and B are collected from the culture. This is a process for producing macrospherides A and B, which are novel cell adhesion inhibitors.
[0006]
[Means for Solving the Problems]
The present inventors isolated strains from various soils for the purpose of searching for new physiologically active substances, and as a result of continuing various searches for metabolites produced by them, newly collected soils in Izu Town, Shizuoka Prefecture It was found that a substance inhibiting the adhesion between human umbilical vein endothelial cells (HUVEC) and human myeloid leukemia (HL-60) cells was produced in the culture of strain FO-5050 isolated from
[0007]
Subsequently, an effective substance that inhibits adhesion between human umbilical vein endothelial cells (HUVEC) and human myeloid leukemia (HL-60) cells was separated and purified from the culture. As a result, macrospheride having the following physicochemical properties was obtained. Since the substances A and B have not been known at all, this substance has been called macrospherides. Further, macrospherides having the physicochemical properties described below are obtained by culturing a production bacterium belonging to the genus Microsphaeropsis in a medium, accumulating the macrospherides in the culture, and Collect rides.
[0008]
As a strain used for using the macrospherides of the present invention, it belongs to the genus Microsphaeropsis, for example, the conidia incompletely isolated from the present inventors, the genus Microspheropsis Microsphaeropsis sp. FO-5050 strain belonging to the above is an example of the strain most effectively used in the present invention, and the bacteriological properties of the strain are as follows.
[0009]
I. Morphological properties It grows well on potato / glucose agar, yeast extract / solvable starch agar (YpSs), etc., and the formation of stalk shell is also good. When the colonies grown on the potato / glucose agar medium are observed with a microscope, the hyphae are transparent and have partition walls, and the formation of stalk shells is good. The stalk shell is black, rarely covered with mycelia, white, spherical, sometimes with a pronounced pore or neck and semi-projecting on the medium. The size is 200-250 μm, and after maturation, black spore mud comes out from the top hole. The shell wall tissue is dark brown. The conidia are light brown, oval to oval, single bubbles and have a size of 4.5 to 5.5 μm × 3.8 μm.
[0010]
II. Table 1 shows the results of macroscopic observation when cultured on various culture media at 25 ° C for 14 days. In each medium, formation of a secretory solution and microbial nuclei accompanying the growth of the bacterium was not observed.
[0011]
[Table 1]

[0012]
III. Physiological and ecological properties 1) The optimum growth conditions for this strain are pH 4-10 in the GP liquid medium, and the optimum growth temperature is 15-30 ° C. in the YpSs agar medium.
2) The growth range of this strain is pH 3 to 10 in the GP liquid medium, and the growth temperature is 7 to 35 ° C. in the YpSs agar medium.
3) Distinguishing between aerobic and anaerobic: aerobic
As a result of attempting comparison with known strains based on the above morphological observation, culture and physiological properties, this strain was identified as a strain belonging to the genus Microspheropsis, The product was named as Microsphere spiss FO-5050. This strain is deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology as Microspheropsis SP FO-5050. The deposit date is February 20, 1995, and the deposit number is FERM P-14776.
[0014]
Although macrospherides producing bacteria have been described as preferred strains of the present invention, the bacteriological properties as general properties of filamentous fungi are very easily mutated, are not constant, and are performed naturally or normally. It is a well-known fact that mutation is caused by ultraviolet irradiation, X-ray irradiation or artificial mutation means using mutant derivatives such as N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfonate, etc. Any strain that belongs to the genus Microspheropsis and has the ability to produce macrospherides, including artificial mutants as well as natural mutants, can be used in the present invention.
[0015]
In addition, strains mutated by cell engineering such as cell fusion and gene manipulation are also included as macrospherides producing bacteria of the present invention. In the present invention, first, macrospherides-producing bacteria belonging to the genus Microspheropsis are cultured in an appropriate medium. In culturing the present bacterium, a usual method for culturing filamentous fungi is generally applied. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and an inorganic salt, if necessary, is used.
[0016]
As the carbon source that can be assimilated, glucose, sugar dense, starch, dextrin, cellulose, corn steep liquor, glycerin, organic acid and the like are used alone or in combination. Digestible nitrogen sources include, for example, commercially available peptone, meat extract, yeast extract, dry yeast, soy flour, corn steep liquor, cottonseed flour, casein, soy proteolysate, amino acids, urea and other organic materials Inorganic nitrogen compounds such as nitrogen sources, nitrates, and ammonium salts are used alone or in combination.
[0017]
Furthermore, inorganic salts such as sodium salt, potassium salt, calcium salt, magnesium salt and phosphate are added as necessary. Furthermore, micronutrients, growth promoting substances, and precursors that promote the growth of the present bacterium and the production of macrospherides may be appropriately added to the medium as necessary.
[0018]
Cultivation is usually carried out under aerobic conditions such as shaking or aeration-agitation culture. Industrially, deep aeration stirring culture is preferable. It is preferable that the culture is carried out at a neutral pH. The culture temperature may be 20 to 37 ° C, but it is usually kept at 24 to 30 ° C, preferably around 27 ° C. In the case of liquid culture, when the culture is usually performed for 3 to 6 days, macrospherides are produced and accumulated. Preferably, the culture may be terminated when the accumulated amount of macrospherides during the culture reaches a maximum.
[0019]
The culture conditions such as the culture composition, the liquidity of the medium, the culture temperature, the stirring speed, and the aeration rate should be appropriately adjusted and selected so as to obtain preferable results according to the type of strain used and external conditions. Needless to say. When there is foaming in liquid culture, antifoaming agents such as silicon oil, vegetable oil, and surfactant may be used as appropriate.
[0020]
The active substance accumulated in the culture thus obtained is contained in the culture filtrate or the cultured cells. If necessary, filter the culture filtrate by adding a filter aid, such as Celite, High Flow Supercell, etc., or centrifuge to separate the culture filtrate and cells, and extract the culture filtrate and cells with organic solvent. It is advantageous to collect macrospherides from a mixture of concentrated products.
[0021]
Moreover, it can extract with a non-hydrophilic organic solvent as it is, without isolate | separating a culture filtrate and a microbial cell. Further, when the content of macrospherides is extremely high in either the culture filtrate or the microbial cells, it may be extracted from the larger one. In order to separate and purify macrospherides from cultured cells, it is usual to extract a mixture of the culture filtrate and the organic solvent extract of the cells or each with a non-immersive solvent such as ethyl acetate or chloroform. By doing so, macrospherides are dissolved in an organic solvent.
[0022]
The organic solvent layer thus obtained is dehydrated by adding various dehydrating agents such as anhydrous sodium sulfate and bead gel as necessary, and then the organic solvent is distilled off under reduced pressure. In this concentration operation, macrospherides are stable substances, but it is usually preferable that the heating temperature be 50 ° C. or lower. Macrospherides can be precipitated by adding an organic solvent such as hexane or petroleum ether to the residue.
[0023]
After the obtained precipitate is washed several times with hexane or the like, a crude product of macrospherides can be collected by suction filtration or centrifugation. In order to further refine this crude product, there are many means that utilize the difference in solubility between macrospherides and congested materials, the difference in the two-liquid correlation distribution that does not mix, and the difference in the adsorption power to various adsorbents. In particular, chromatography is an effective method for purifying macrospherides.
[0024]
Effective chromatography for purification of macrospherides includes adsorption chromatography using silica gel, alumina, activated carbon cellulose, adsorption resin such as hydroxyapatite HP-20, reverse phase partition using silanized silica gel, octadecyl silanized silica gel, etc. Examples thereof include gel filtration chromatography based on molecular sieve using chromatography, Sephadex LH-20, Toyopearl (trade name, manufactured by Tosoh Corporation) and the like.
[0025]
Macrospherides can be separated and purified by using these means such as chromatography, electrophoresis, countercurrent distribution, and ultrafiltration alone or in combination or repeatedly in any order. For example, the above crude product is dissolved in a small amount of chloroform, adsorbed onto silica gel, and subjected to column chromatography using a chloroform-acetone mixed solution. The active fraction is concentrated under reduced pressure, and then dissolved in a small amount of methanol. The macrosphere can be separated and purified by gel filtration chromatography based on molecular sieve.
[0026]
Next, the physicochemical properties of the present macrospherides will be described below.
[1] Cell adhesion inhibitor Macrospheride A
(1) Property: colorless needle crystal (2) Elemental composition: C ₁₆ H ₂₂ O ₈ (according to high resolution mass spectrum)
(3) Melting point: 141-142 ° C. (chloroform)
(4) Specific rotation: [α] _D ²³ + 84.1 ° (c = 0.59, MeOH)
(5) Molecular weight: 342 (according to fast atom bombardment (FAB) mass spectrum)
[0027]
(6) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 1, and shows a characteristic absorption maximum in the vicinity of 207 (log ε = 4.21) nm. (7) Infrared absorption spectrum : Infrared absorption spectrum measured by KBr method is as shown in FIG. 2 and has an absorption band at 1713, 3428 cm ⁻¹ (8) Solubility in solvent: acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, Soluble in chloroform, hardly soluble in water 【0028】
(9) Color reaction: positive to sulfuric acid, phosphomolybdic acid, iodine, P-anisaldehyde (10) Nuclear magnetic resonance spectrum: ¹ H-NMR spectrum (deuterated chloroform solution) measured using JEOL EX-270 NMR spectrometer Middle, 270 MHz) and ¹³ C-NMR spectrum (in deuterated chloroform solution, 67.8 MHz) are as shown in FIGS. 3 and 4, respectively. The chemical shifts of hydrogen and carbon are as shown in Table 2 below.
[0029]
[Table 2]

[0030]
[2] Cell adhesion inhibitor Macrospheride B
(1) Properties: colorless plate crystals (2) Elemental composition: C ₁₆ H ₂₀ O ₈ (according to high resolution mass spectrum)
(3) Melting point: 148 to 150 ° (chloroform)
(4) Specific rotation: [α] _D ²³ + 4.1 ° (c = 0.985, MeOH)
(5) Molecular weight: 340 (according to fast atom bombardment (FAB) mass spectrum)
[0031]
(6) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in FIG. 5, and shows a characteristic absorption maximum in the vicinity of 210 (log ε = 4.15) nm. (7) Infrared absorption spectrum : Infrared absorption spectrum measured by the KBr method is as shown in FIG. 6 and has absorption bands at 3458, 1743, 1726, 1705 cm ^−1.
(8) Solubility in solvents: Soluble in acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, chloroform, hardly soluble in water (9) Color reaction: sulfuric acid, phosphomolybdic acid, iodine, P-anisaldehyde Positive for [0033]
(10) Nuclear magnetic resonance spectrum: ¹ H-NMR spectrum (270 MHz in deuterated chloroform solution) and ¹³ C-NMR spectrum (in deuterated chloroform solution, 67.8 MHz) measured using JEOL EX-270 NMR spectrometer are These are as shown in FIGS. 7 and 8, respectively. The chemical shifts of hydrogen and carbon are as shown in Table 3 below.
[0034]
[Table 3]

[0035]
Evaluation of Cell Adhesion Inhibitory Ability Using Human Vascular Endothelial Cells Cell adhesion inhibitory activity was tested using the binding ability of human myeloid leukemia cells (HL-60) to human umbilical vein endothelial cells (HUVEC) as an index.
2 × 10 ⁴ HUVECs suspended in MCDB107 medium containing 10% fetal bovine serum (FBS), heparin 100 U / ml, ECGF 50 μg / ml are seeded in each hole of a 96-well microplate, and 37 in a CO ₂ incubator. Incubate at ℃ for 24 hours.
[0036]
10 ng of tumor necrosis factor (TNF) was then added to stimulate HUVEC. After 5 hours, the specimen and fluorescently labeled HL-60 were added and incubated for 30 minutes. After removing non-adherent cells, the cell adhesion rate was calculated by measuring the fluorescence intensity of each hole. The results are shown in Table 4.
[0037]
[Table 4]

[0038]
【The invention's effect】
As described above, macrospherides A and B according to the present invention are expected to have an effect on diseases considered to be deeply involved in cell adhesion, such as cancer metastasis, chronic inflammation such as arthritis, asthma, allergic diseases and the like. .
[0039]
【Example】
EXAMPLES Next, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
In a test tube (φ2 × 20 cm), glucose 2.0%, yeast extract 0.2%, magnesium sulfate heptahydrate 0.05%, polypeptone 0.5%, potassium dihydrogen phosphate 0.1%, agar Sterilize 0.1% (pH 5.6 to 5.8), 10 ml, yeast extract 0.4%, soluble starch 1.5%, potassium hydrogen phosphate 0.1%, magnesium sulfate 7 water One platinum from the slope culture of Microspheropsis sp. FO-5050 (FERM P-14476) cultured at 27 ° C. for 6 days on an agar slope medium containing 0.05% Japanese and 2.0% agar (pH 6.0) Ears were inoculated and cultured at 27 ° C. for 72 hours with shaking to obtain seed mothers.
[0040]
Next, in a 500 ml Erlenmeyer flask, soluble starch 3.0%, glycerol 1.0%, kinaco 2.0%, dry yeast 0.3%, potassium chloride 0.3%, calcium carbonate 0.2%, magnesium sulfate -After seeding and sterilizing 100 ml of liquid production medium (pH 6.5) containing 0.05% heptahydrate and 0.05% potassium dihydrogen phosphate, seed 1 obtained by the above method 0.0 ml was transplanted aseptically into the above Erlenmeyer flask, and cultured with shaking at 27 ° C. for 120 hours on a rotary shaker rotating at 220 rpm.
[0041]
The obtained culture broth was divided into a liquid fraction and a fungus body fraction, and the ethyl acetate extract fraction was obtained by liquid separation after adding ethyl acetate and stirring sufficiently. For the cell fraction, methanol was added thereto, and the mixture was sufficiently stirred. The obtained methanol fraction was concentrated under reduced pressure to distill off the methanol, followed by extraction with ethyl acetate. The ethyl acetate fractions obtained from both the liquid and bacterial cell fractions were combined, and 379.4 mg of an ethyl acetate extract fraction was obtained by concentration under reduced pressure.
[0042]
This ethyl acetate extract fraction was adsorbed on two thin-layer silica gels (20 × 20 cm, thickness 0.5 mm, manufactured by Merck) and developed with a chloroform-methanol mixed solvent (9: 1). A band showing an orange-yellow fluorescence under irradiation with an ultraviolet lamp (254, 366 nm) at an Rf value of about 0.5 was fractionated and eluted with a chloroform-methanol mixed solvent (1: 1). The obtained eluted fraction was concentrated under reduced pressure to obtain 18.3 mg of crude substance I containing macrospherides.
[0043]
This crude substance I was dissolved in a small amount of methanol, subjected to high performance liquid chromatography (Senshu Pack Pegacil, φ20 × 250 mm), and the active fraction was fractionated using a 40% acetonitrile solution as a mobile phase to prepare Macrospheride A And 10.2 mg of pure product and 5.2 mg of macrospheride B were obtained.
[Brief description of the drawings]
FIG. 1 is an ultraviolet absorption spectrum of Macrospheride A.
FIG. 2 is an infrared absorption spectrum of Macrospheride A.
FIG. 3 is a ¹ H-nuclear magnetic resonance spectrum of Macrospheride A.
FIG. 4 is a ¹³ C-nuclear magnetic resonance spectrum of Macrospheride A.
FIG. 5 is an ultraviolet absorption spectrum of Macrospheride B.
6 is an infrared absorption spectrum of Macrospheride B. FIG.
7 is a ¹ H-nuclear magnetic resonance spectrum of Macrospheride B. FIG.
8 is a ¹³ C-nuclear magnetic resonance spectrum of macrospheride B. FIG.

Claims (4)

Cell adhesion inhibitors Macrospheride A and B having the following physicochemical properties:
[1] Cell adhesion inhibitor Macrospheride A
(1) Property: colorless needle crystal (2) Elemental composition: C ₁₆ H ₂₂ O ₈ (according to high resolution mass spectrum)
(3) Melting point: 141-140 ° C. (chloroform)
(4) Specific rotation [α] _D ²³ + 84.1 ° (c = 0.59, MeOH)
(5) Molecular weight: 342 (according to fast atom bombardment (FAB) mass spectrum)
(6) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol shows a characteristic absorption maximum in the vicinity of 207 (log ε = 4.21) nm. (7) Infrared absorption spectrum: Infrared measured by KBr method Absorption spectrum has absorption bands at 1713 and 3428 cm ⁻¹ (8) Solubility in solvents: Soluble in acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, chloroform, hardly soluble in water (9) Coloration Reaction: Positive for sulfuric acid, phosphomolybdic acid, iodine, P-anisaldehyde (10) ¹ H-nuclear magnetic resonance spectrum : ¹ H-shift (J value) measured in deuterated chloroform is 6.87 (1H, dd , J = 15.5, 3.3), 6.88 (1H, dd, J = 15.5, 3.3), 6.03 (1H, dd, J = 15.5, 1.8) ), 6.04 (1H, dd, J = 15.5, 1.5), 4.97 (1H, q, J = 6.6), 4.25 (1H, br.s), 4.86 (1H, q, J = 6.6), 4.13 (1H, br.s), 5.38 (1H, m), 2.60 (1H, dd, J = 8.3, 4.3) 1.33 (3H, d, J = 6.3), 1.45 (3H, d, J = 6.6), 1.37 (3H, d, J = 6.6), 3.15 ( 1H, br.s), 2.85 (1H, br.s)
(11) ¹³ C-nuclear magnetic resonance spectrum : ¹³ C-shift measured in deuterated chloroform is 170.2 (s), 165.8 (s), 164.7 (s), 146.2 (d). 145.2 (d), 122.7 (d), 122.2 (d), 74.8 (d), 74.7 (d), 73.9 (d), 73.0 (d), 67.7 (d), 40.9 (t), 19.6 (q), 17.9 (q), 17.8 (q), -OH, -OH
[2] Cell adhesion inhibitor Macrospheride B
(1) Properties: colorless plate crystals (2) Elemental composition: C ₁₆ H ₂₀ O ₈ (according to high resolution mass spectrum)
(3) Melting point: 148 to 150 ° C. (chloroform)
(4) Specific rotation [α] _D ²³ + 4.1 ° (c = 0.985, MeOH)
(5) Molecular weight: 340 (according to fast atom bombardment (FAB) mass spectrum)
(6) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol shows a characteristic absorption maximum in the vicinity of 210 (log ε = 4.15) nm. (7) Infrared absorption spectrum: Infrared measured by KBr method The absorption spectrum has absorption bands at 3458 , 1743, 1726, and 1705 cm ⁻¹ (8) Solubility in solvents: soluble in acetone, ethyl acetate, ethyl ether, hexane, methanol, ethanol, chloroform, hardly soluble in water ( 9) Color reaction: positive to sulfuric acid, phosphomolybdic acid, iodine, P-anisaldehyde (10) ¹ H-nuclear magnetic resonance spectrum : ¹ H-shift (J value) measured in deuterated chloroform is 6.94. (1H, dd, J = 15.8, 3.6), 6.73 (1H, d, J = 15.8), 7.03 (1H, d <J = 15.8) ), 6.08 (1H, dd, J = 15.8, 3.0), 5.08 (2H, m), 5.08 (2H, m), 4.32 (1H, br.s), 5.45 (1H, m), 2.83 (1H, dd, J = 16.2, 10.9), 2.64 (1H, dd, J = 16.2, 3.0), 1.36 (3H, d, J = 6.6), 1.49 (1H, dd, J = 6.6), 1.43 (1H, dd, J = 6.9), 3.44 (br.s)
(11) ¹³ C-nuclear magnetic resonance spectrum : ¹³ C-shift measured in deuterated chloroform is 196.2 (s), 170.4 (s), 165.2 (s), 164.3 (s) 144.5 (d), 132.6 (d), 132.1 (d), 122.4 (d), 76.5 (d), 75.7 (d), 74.6 (d), 67.7 (d), 40.6 (t), 19.7 (q), 17.8 (q), 16.0 (q), -OH The microorganism having the ability to produce macrospherides A and B according to claim 1 belonging to the genus Microspheropsis is cultured in a medium to accumulate macrospherides A and B in the culture, from which macross A method for producing a novel cell adhesion inhibitor Macrospheride A and B, which comprises collecting ferrides A and B. The microorganism belonging to the genus Microspheropsis and having the ability to produce macrospherides A and B according to claim 1 is Microspheropsis sp. FO-5050 ( FERM P-147 76) Item 3. The production method according to Item 2. 2. Microspheres sp. FO-5050 (FERM P-14776), which belongs to the genus Microspheropsis and has the ability to produce macrospherides A and B according to claim 1 .

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